SummaryWhile reports suggest a single dose of senolytics may improve vasomotor function, the structural and functional impact of long‐term senolytic treatment is unknown. To determine whether long‐term senolytic treatment improves vasomotor function, vascular stiffness, and intimal plaque size and composition in aged or hypercholesterolemic mice with established disease. Senolytic treatment (intermittent treatment with Dasatinib + Quercetin via oral gavage) resulted in significant reductions in senescent cell markers (TAF + cells) in the medial layer of aorta from aged and hypercholesterolemic mice, but not in intimal atherosclerotic plaques. While senolytic treatment significantly improved vasomotor function (isolated organ chamber baths) in both groups of mice, this was due to increases in nitric oxide bioavailability in aged mice and increases in sensitivity to NO donors in hypercholesterolemic mice. Genetic clearance of senescent cells in aged normocholesterolemic INK‐ATTAC mice phenocopied changes elicited by D+Q. Senolytics tended to reduce aortic calcification (alizarin red) and osteogenic signaling (qRT–PCR, immunohistochemistry) in aged mice, but both were significantly reduced by senolytic treatment in hypercholesterolemic mice. Intimal plaque fibrosis (picrosirius red) was not changed appreciably by chronic senolytic treatment. This is the first study to demonstrate that chronic clearance of senescent cells improves established vascular phenotypes associated with aging and chronic hypercholesterolemia, and may be a viable therapeutic intervention to reduce morbidity and mortality from cardiovascular diseases.

Abstract-In cultured endothelial cells, the antioxidant, L-ascorbic acid (vitamin C), increases nitric oxide synthase (NOS) enzyme activity via chemical stabilization of tetrahydrobiopterin. Our objective was to determine the effect of vitamin C on NOS function and tetrahydrobiopterin metabolism in vivo. Twenty-six to twenty-eight weeks of diet supplementation with vitamin C (1%/kg chow) significantly increased circulating levels of vitamin C in wild-type (C57BL/6J) and apolipoprotein E (apoE)-deficient mice. Measurements of NOS enzymatic activity in aortas of apoE-deficient mice indicated a significant increase in total NOS activity. However, this increase was mainly due to high activity of inducible NOS, whereas eNOS activity was reduced. Significantly higher tetrahydrobiopterin levels were detected in aortas of apoE-deficient mice. Long-term treatment with vitamin C restored endothelial NOS activity in aortas of apoE-deficient mice, but did not affect activity of inducible NOS. In addition, 7,8-dihydrobiopterin levels, an oxidized form of tetrahydrobiopterin, were decreased and vascular endothelial function of aortas was significantly improved in apoE-deficient mice. Interestingly, vitamin C also increased tetrahydrobiopterin and NOS activity in aortas of C57BL/6J mice. In contrast, long-term treatment with vitamin E (2000 U/kg chow) did not affect vascular NOS activity or metabolism of tetrahydrobiopterin. In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of tetrahydrobiopterin and restoration of eNOS enzymatic activity. Key Words: tetrahydrobiopterin Ⅲ nitric oxide synthase Ⅲ nitric oxide Ⅲ antioxidants Ⅲ superoxide anion N itric oxide (NO) is a potent vasodilator and plays a key role in control of the cardiovascular system. 1 NO is mainly formed in endothelial cells from L-arginine by oxidation of its terminal guanidino-nitrogen, 2 requiring the cofactors NADPH, (6R)-5,6,7,8-tetrahydrobiopterin (BH 4 ), FAD, FMN, heme, and Zn 2ϩ . 3,4 The formation of NO occurs via endothelial NO-synthase (eNOS) which is expressed constitutively. 5,6 Relaxations in response to the abluminal release of endothelium-derived NO are associated with stimulation of soluble guanylyl cyclase (sGC) and in turn formation of cyclic guanosine 3Ј,5Ј-monophosphate (cGMP) in vascular smooth muscle cells. 7 Inducible NOS (iNOS) enzyme can be expressed in vascular smooth muscle cells, endothelium, and macrophages. This enzyme activity is Ca 2ϩ -independent and produces large amounts of NO; it is induced by cytokines such as interleukin 1␤ and tumor necrosis factor-␣ and hence is activated in atherosclerosis and inflammatory processes. 8 -11 BH 4 is an essential cofactor required for activity of all NOS isoforms. 4,12 During activation of NOS, BH 4 is needed for allosteric and redox activation of its enzymatic activity. 4,13 Accumulating evidence suggests that alterations in the NO pathway, such as increased NO decomposition by superoxide anion (O 2 Ϫ ) or altered NOS...

Abstract-Endothelium-dependent relaxations mediated by NO are impaired in a mouse model of human atherosclerosis.Our objective was to characterize the mechanisms underlying endothelial dysfunction in aortas of apolipoprotein E (apoE)-deficient mice, treated for 26 to 29 weeks with a lipid-rich Western-type diet. Aortic rings from apoE-deficient mice showed impaired endothelium-dependent relaxations to acetylcholine (10 Ϫ9 to 10 Ϫ5 mol/L) and Ca 2ϩ ionophore (10 Ϫ9 to 10 Ϫ6 mol/L) and endothelium-independent relaxations to diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10 Ϫ10 to 10 Ϫ5 mol/L) compared with aortic rings from C57BL/6J mice (PϽ0.05). By use of confocal microscopy of an oxidative fluorescent probe (dihydroethidium), increased superoxide anion (O 2 Ϫ ) production was demonstrated throughout the aortic wall but mainly in smooth muscle cells of apoE-deficient mice. CuZn-superoxide dismutase (SOD) and Mn-SOD protein expressions were unaltered in the aorta exposed to hypercholesterolemia. A cell-permeable SOD mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (10 Ϫ5 mol/L), reduced O 2 Ϫ production and partially normalized relaxations to acetylcholine and DEA-NONOate in apoE-deficient mice (PϽ0.05). [ 14 C]L-Citrulline assay showed a decrease of Ca 2ϩ -dependent NOS activity in aortas from apoE-deficient mice compared with C57BL/6J mice (PϽ0.05), whereas NO synthase protein expression was unchanged. In addition, cGMP levels were significantly reduced in the aortas of apoE-deficient mice (PϽ0.05). Our results demonstrate that in apoE-deficient mice on a Western-type fat diet, impairment of endothelial function is caused by increased production of O 2 Ϫ and reduced endothelial NO synthase enzyme activity. Key Words: endothelium Ⅲ nitric oxide Ⅲ superoxide anion Ⅲ apolipoprotein E Ⅲ atherosclerosis A therosclerosis is a chronic process, which can be triggered by cardiovascular risk factors such as hypercholesterolemia, aging, hypertension, and diabetes mellitus. 1 Endothelium-derived vasoactive factors play an important regulatory role in vascular homeostasis and pathogenesis of atherosclerosis because of the strategic position of the endothelium between the vascular smooth muscle cells (VSMCs) and the circulating blood. 2,3 NO is a potent vasodilator that is formed in endothelial cells from L-arginine by endothelial NO synthase (eNOS), which is constitutively expressed. 4 -6 NO production is activated by the stimulation of cell surface receptors or by mechanical forces such as shear stress. 7,8 Accumulating evidence suggests that alterations in the NO pathway play a central role in endothelial dysfunction induced by hypercholesterolemia. This may be of major importance inasmuch as NO can substantially inhibit several components of the atherogenic process, such as VSMC contraction and proliferation, platelet aggregation, and monocyte adhesion. 9,10 Previous studies identified 3 mechanisms responsible for reduced bioavailability of NO in arteries exposed to hypercholester...

Background and Purpose-Circulating endothelial progenitor cells (EPCs) play an important role in repair of injured vascular endothelium and neovascularization. The present study was designed to determine the effect of EPCs transplantation on the regeneration of endothelium and recovery of endothelial function in denuded carotid arteries. Methods-Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium for 7 days, yielding EPCs. A rabbit model of common carotid artery denudation by passage of a deflated balloon catheter was used to evaluate the effects of EPCs on endothelial regeneration and vasomotor function. Immediately after denudation, autologous EPCs (

Nitric oxide (NO), synthesized from Larginine by NO synthases (NOS), plays an essential role in the regulation of cerebrovascular tone. Adenoviral vectors have been widely used to transfer recombinant genes to different vascular beds. To determine whether the recombinant endothelial NOS (eNOS) gene can be delivered in vivo to the adventitia of cerebral arteries and functionally expressed, a replication-incompetent adenoviral vector encoding eNOS gene (AdCMVNOS) or ␤-galactosidase reporter gene (AdCMVLacZ) was injected into canine cerebrospinal f luid (CSF) via the cisterna magna (final viral titer in CSF, 10 9 pfu͞ml). Adventitial transgene expression was demonstrated 24 h later by ␤-galactosidase histochemistry and quantification, eNOS immunohistochemistry, and Western blot analysis of recombinant eNOS. Electron microscopy immunogold labeling indicated that recombinant eNOS protein was expressed in adventitial fibroblasts. In AdCMVNOS-transduced arteries, basal cGMP production and bradykinin-induced relaxations were significantly augmented when compared with AdCMVLacZ-transduced vessels (P < 0.05). The increased receptormediated relaxations and cGMP production were inhibited by eNOS inhibitors. In addition, the increase in cGMP production was reversed in the absence of calcium, suggesting that the increased NO production did not result from inducible NOS expression. The present study demonstrates the successful in vivo transfer and functional expression of recombinant eNOS gene in large cerebral arteries. It also suggests that perivascular eNOS gene delivery via the CSF is a feasible approach that does not require interruption of cerebral blood f low.

We investigated a transgenic mouse model of sickle cell disease, homozygous for deletion of mouse beta-globin and containing transgenes for human beta(S) and beta(S-antilles) globins linked to the transgene for human alpha-globin. In these mice, basal cGMP production in aortic rings is increased, whereas relaxation to an endothelium-dependent vasodilator, A-23187, is impaired. In contrast, aortic expression of endothelial nitric oxide synthase (NOS) is unaltered in sickle mice, whereas expression of inducible NOS is not detected in either group; plasma nitrate/nitrite concentrations and NOS activity are similar in both groups. Increased cGMP may reflect the stimulatory effect of peroxides (an activator of guanylate cyclase), because lipid peroxidation is increased in aortae and in plasma in sickle mice. Despite increased vascular cGMP levels in sickle mice, conscious systolic blood pressure is comparable to that of aged-matched controls; sickle mice, however, evince a greater rise in systolic blood pressure in response to nitro-L-arginine methyl ester, an inhibitor of NOS. Systemic concentrations of the vasoconstrictive oxidative product 8-isoprostane are increased in sickle mice. We conclude that vascular responses are altered in this transgenic sickle mouse and are accompanied by increased lipid peroxidation and production of cGMP; we suggest that oxidant-inducible vasoconstrictor systems such as isoprostanes may oppose nitric oxide-dependent and nitric oxide-independent mechanisms of vasodilatation in this transgenic sickle mouse. Destabilization of the vasoactive balance in the sickle vasculature by clinically relevant states may predispose to vasoocclusive disease.

Oxidative stress has been implicated as an important mechanism of vascular endothelial dysfunction induced by aging. Previous studies suggested that tetrahydrobiopterin (BH 4), an essential cofactor of endothelial NO synthase, could be a molecular target for oxidation. We tested the hypothesis that oxidative stress, in particular oxidation of BH 4, may contribute to attenuation of endothelium-dependent relaxation in aged mice. Vasomotor function of isolated carotid arteries was studied using a video dimension analyzer. Vascular levels of BH 4 and its oxidation products were measured via HPLC. In aged mice (age, 95 Ϯ 2 wk), endothelium-dependent relaxation to ACh (10 Ϫ5 to 10 Ϫ9 M) as well as endothelium-independent relaxation to the NO donor diethylammo-Ϫ5 to 10 Ϫ9 M) were significantly reduced compared with relaxation detected in young mice (age, 23 Ϯ 0.5 wk). Incubation of aged mouse carotid arteries with the cell-permeable SOD mimetic Mn(III)tetra(4-benzoic acid)porphyrin chloride normalized relaxation to ACh and DEA-NONOate. Furthermore, production of superoxide anion in aorta and serum levels of amyloid P component, which is the murine analog of C-reactive protein, was increased in old mice. In aorta, neither the concentration of BH 4 nor the ratio of reduced BH4 to the oxidation products were different between young and aged mice. Our results demonstrate that in mice, aging impairs relaxation mediated by NO most likely by increased formation of superoxide anion. Oxidation of BH 4 does not appear to be an important mechanism underlying vasomotor dysfunction in aged mouse arteries. endothelial dysfunction; nitric oxide; superoxide anion; reactive oxygen species; C-reactive protein AGING IS A RISK FACTOR for vascular disease; however, the role of aging, either as a process or as the result of longer exposure to other risks, is not well defined (16,17). In murine vascular tissue, the age-dependent changes in vasomotor function have not been characterized. Studies on rats have shown impairment of endothelium-dependent relaxation due to increased production of superoxide anions, but the source of the superoxide anions has also not been characterized (16,30). Reactive oxygen species (ROS) have been implicated in endothelial dysfunction associated with aging, hypertension, hypercholesteremia, diabetes, and cigarette smoking (3). ROS can interfere with endothelium-dependent relaxation particularly by the scavenging of NO by superoxide anion (O 2 Ϫ ⅐; Refs. 1, 3, 34).

II induces vasoconstriction, at least in part, by stimulating NADPH oxidase and generating reactive oxygen species. ANG II also induces heme oxygenase activity, and bilirubin, a product of such activity, possesses antioxidant properties. We hypothesized that bilirubin, because of its antioxidant properties, may reduce the pressor and prooxidant effects of ANG II. Our in vivo studies used the hyperbilirubinemic Gunn rat which is deficient in the enzyme uridine diphosphate glucuronosyl transferase, the latter enabling the excretion of bilirubin into bile. ANG II (0.5 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) or saline vehicle was administered by osmotic minipump to control and Gunn rats for 4 wk. The rise in systolic blood pressure induced by ANG II, as observed in control rats, was markedly reduced in Gunn rats, the latter ϳ50% less at 3 and 4 wk after the initiation of ANG II infusion. The chronic administration of ANG II also impaired endothelium-dependent relaxation responses in control rats but not in Gunn rats. As assessed by the tetrahydrobiopterin/dihydrobiopterin ratio, ANG II induced oxidative stress in the aorta in control rats but not in Gunn rats. Heightened generation of superoxide anion in aortic rings in ANG II-infused rats and by vascular smooth muscle cells exposed to ANG II was normalized by bilirubin in vitro. We conclude that the pressor and prooxidant effects of ANG II are attenuated in the hyperbilirubinemic Gunn rat, an effect which, we speculate, may reflect, at least in part, the scavenging of superoxide anion by bilirubin. NADPH oxidase; heme oxygenase; nitric oxide; endothelial function ANG II ACTIVATES THE NADPH oxidase enzyme system and promotes the generation of superoxide anion and other reactive oxygen species (15,25,36,40,43). Such stimulation of superoxide anion contributes significantly to the vasoconstrictive and pressor effects of ANG II, as demonstrated by studies that use antioxidants including those that scavenge the superoxide anion (25,36,40,43). Indeed, it is generally accepted that systemic hypertension induced by the chronic administration of ANG II in rodents arises, at least in part, from the generation of superoxide anion as NADPH oxidase is activated by ANG II (25,36,40,43).Our prior studies as well as the studies of others demonstrate that systemic administration of ANG II leads to marked upregulation of heme oxygenase-1 (HO-1) (4, 15, 49). HO is the rate-limiting enzyme in the degradation of heme, enabling the conversion of heme to biliverdin, in the course of which carbon monoxide evolves and iron is liberated from the heme ring; subsequently, biliverdin is converted to bilirubin (2,3,31,32,39). The HO-1 isoform is rapidly induced by diverse stimuli and often confers a cytoprotective, anti-inflammatory, and vasorelaxant effect (2,3,31,32,39). The induction of HO-1 following the administration of ANG II likely exerts a countervailing, vasorelaxant effect: prior upregulation of HO-1 reduces the rise in systemic blood pressure induced by ANG II, whereas inhibition of HO activity exace...